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Rice ERF OsEATB restricts GA biosynthesis - Plant Physiology

Rice ERF OsEATB restricts GA biosynthesis - Plant Physiology

grew well on SD/Trp-Leu-

grew well on SD/Trp-Leu- medium, but not on SD/Trp-Leu-His-/50mM 3-AT medium. The band corresponding to the GCC-box-containing labeled probe and purified His fusion recombinant OsEATB protein complex showed a marked mobility shift compared with the free probe band (Fig. 1D). And mGCC-box-containing labeled fragment was served as competitor. These results demonstrated that OsEATB could specifically bind to the GCC-box. If OsEATB acts as a transcription factor, then its NLS should localize it to the nucleus. To confirm the subcellular localization of OsEATB, we fused the coding sequence of OsEATB with that of green fluorescent protein (GFP) under the control of the 35S promoter (35S:OsEATB:GFP) and delivered the construct into onion epidermal cells by particle bombardment (Fig. 1E). The OsEATB protein localized the GFP signal to the nucleus (Fig. 1F). We investigated the tissue expression pattern of OsEATB by reverse transcriptase (RT)-PCR using rice total mRNA from roots, culms, leaves, and young panicles as the template. The results showed that OsEATB mRNA is expressed constitutively in these four tissues and is expressed at higher levels in roots and leaves than in culms and young panicles (Fig. 1G). Expression of OsEATB in transgenic rice lines To investigate the function of OsEATB, we introduced the plasmid 35S:OsEATB containing the OsEATB gene (Fig. 2A) into O. sativa L. subsp. Indica var. 9311. We produced transgenic plants over-expressing the sense strand of OsEATB. 35S:OsEATB transformants were screened on antibiotic selection medium containing hygromycin. The transgenic plants were checked by PCR using genomic DNA as the template and specific primers (as described in materials and methods). Eight independent transformants (T0), regenerated from hygromycin-resistant calli, were shown to contain OsEATB. Eight PCR-positive T1 lines (20 individual plants of each line) shared the same dwarf phenotype. Two transgenic lines (401003 and 401006) were selected for Southern hybridization analysis. A single, specific band of OsEATB transgene 8

was observed in 35S:OsEATB transgenic lines 401003 and 401006, respectively (Fig. 2B). These results showed that OsEATB was integrated into the rice genome. The expression level of the OsEATB gene was examined in 3-leaf-stage transgenic plants and control plants by real-time PCR analyses (Fig. 2C) Expression of OsEATB was 9.77-fold greater in the 401006 transgenic plants, and 4.00-fold, 2.83-fold, 2.00-fold greater in the 401003,401005,401007 transgenic plants than in 9311 control plants, respectively. These results were further confirmed by RNA hybridization in situ of the 3-leaf-stage 401003 transgenic plants and 9311 control plants (Fig. 2D). All of these results demonstrated that the OsEATB gene is strongly expressed and genetically transmitted to the next generation. Effect of transgenic OsEATB on rice internode elongation Plant height is not only a decisive factor in plant architecture, but also an important agronomic trait that is directly linked to yield potential (Yang and Hwa, 2008; Huang et al., 1996). The OsEATB transgenic lines showed dwarf phenotypes, indicating that the internodal elongation process was suppressed by OsEATB over-expression. For experiments, we selected two transgenic lines: 401003 and 401006 (20 individual plants of each line). The transgenic plants and 9311 control plants were cultivated in the same conditions and plant height was determined at maturity. The average plant heights of the control, 401003, and 401006 lines were 121, 113, and 106 cm, respectively. Thus, 401003 and 401006 plants were 6.61 and 12.40% shorter than the control, respectively (Fig. 3A, see Supplemental Table S1 online). Furthermore, every elongated internode was shortened, especially the fourth internode, which was shortened by an average of 56.30% (Fig. 3A). OsEATB negatively regulated plant height at every growth stage of rice plants. The transgenic plants showed a dwarf phenotype from the 3-leaf-stage to the mature stage (Fig. 3B, Fig. 6A and 6B). The panicles also showed reduced elongation, demonstrating that 9

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